The present invention relates to a method for enhancing enzyme activity at an elevated temperature by using a substance exhibiting chaperone function.
In general, enzymes exhibit lower activity at a temperature above their optimum temperature than the activity at their optimum temperature. It is also known that their activity is lost when they are exposed to a temperature higher than a certain level. Depending on the kind of enzyme, a temperature at which such heat inactivation occurs may vary. However, most of enzymes having optimum temperature of ordinary temperature are inactivated when heated to around 50xc2x0 C. Enzymes stable at an elevated temperature are also known and such heat-resistant enzymes generally have a higher optimum temperature.
Depending on the conditions where enzymes are used, it is often desirable to use enzymes at an elevated temperature. In such a case, a heat-resistant enzyme as mentioned above is generally used. Examples of such a heat-resistant enzyme include Taq polymerase, which is frequently used for PCR. However, in many cases, a suitable heat-resistant enzyme may not be known, or even if a possible heat-resistant enzyme is known, other conditions to be used may not meet the enzyme.
For example, Superscript II is known as a reverse transcriptase (RNA-dependent DNA polymerase) which can afford a cDNA from a mRNA in vitro. Superscript II is a heat-labile enzyme exhibiting an optimum temperature of 42xc2x0 C. and completely inactivated at a temperature above 50xc2x0 C. within 10 minutes. Although Tth DNA polymerase is an enzyme having heat resistance and reverse transcription activity, it requires manganese ions for exerting the enzyme activity. If cDNAs are produced from mRNAs at a higher temperature using the Tth DNA polymerase, mRNAs are fragmented by manganese ions presented in a reaction system and therefore it becomes difficult to obtain full length cDNAs.
Magnesium ions required by a heat-labile reverse transcriptase such as Superscript II mentioned above may also cause the fragmentation of mRNA in a certain buffer or water at an elevated temperature. According to the present inventor""s researches, manganese ions exhibit stronger fragmentation activity than magnesium ions and control of the fragmentation due to manganese ions is difficult even using a chelating agent.
Taq polymerase is known as an inherently heat-resistant enzyme. However, it shows reduction of activity during 25 to 30 cycles or more generally used in PCR. Therefore, if the reduction of Taq polymerase activity can be prevented, higher amplification effect and higher cycle number can be realized with fewer units of the enzyme.
In some cases, reverse transcription may be required to be performed at a temperature above 50xc2x0 C. for some reasons. For example, in order to obtain full length cDNAs, it is desirable to preform reverse transcription while preventing the formation of secondary structure of mRNAs.
However, a heat-resistant enzyme having reverse transcription activity such as Tth DNA polymerase cannot afford full length cDNAs. Therefore, it is necessary to utilize a currently available reverse transcriptase which is used at an ambient temperature.
Similar situation may be frequently found in other enzymes not only polymerases but also restriction enzymes.
For some enzymes, it has been known that an enzyme exhibiting a higher optimum temperature can be obtained by introducing a mutation through genetic engineering. However, such improvement of heat-resistance is not always possible and has not been known so long as reverse transcriptase concerns.
If an enzyme can exhibit higher activity at a higher temperature, its utility may be enhanced even though it is known as a heat resistant enzyme.
Therefore, the object of the present invention is to provide a method for easily and efficiently improving heat resistance of enzyme to obtain high enzyme activity at an elevated temperature.